Mutant N-acetylglutamate synthase and method for L-arginine production

ABSTRACT

L-arginine is produced using a bacterium belonging to the genus  Escherichia  harboring a mutant N-acetylglutamate synthase in which the amino acid sequence corresponding to positions from 15 to 19 in a wild type N-acetylglutamate synthase is replaced with any one of amino acid sequences of SEQ ID NOS: 1 to 4, and feedback inhibition by L-arginine is desensitized.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to microbiological industry, to the methodof L-arginine production and concerns the using of newfeedback-resistant mutant enzymes in arginine biosynthesis pathway of E.coli arginine-producer strains.

2. Description of the Related Art

The biosynthesis of arginine from glutamate in E. coli cells is carriedout by a series of reactions initiated by the acetylation of glutamateby N-acetylglutamate synthase (NAGS) encoded by argA. This process isregulated via transcription repression of the arg regulon and byfeedback inhibition of NAGS by arginine [Cunin R., et al., Microbiol.Rev., vol.50, p.314-352, 1986]. L-Arginine represses argA expressionwith a ratio greater than 250 and inhibits NAGS activity (Ki=0.02 mm)[Leisinger T., Haas D., J. Biol. Chem., vol.250, p.1690-1693, 1975]. Forenhanced biosynthesis of arginine in E. coli, the feedback-resistant(may be referred to as “fbr”) NAGS enzymes are required.

The feedback-resistant mutants of enzymes can be obtained byspontaneous, chemical or site-directed mutagenesis.

Some argA fbr mutants were isolated and studied. The Serratia marcescenscells carrying the chromosomal fbr argA mutations were unstable and gaverise to argA mutants with reduced activity or with altered affinity forglutamate [Takagi T., et al., J.Biochem. vol.99, p.357-364 1986].

The fbr argA genes from the five E. coli strains with fbr NAGS werecloned and different single-base substitutions in argA genes were foundin each of the fbr NAGS strains and it was revealed that thesubstitutions cause replacing His-15 with Tyr, Tyr-19 with Cys, Ser-54with Asn, Arg-58 with His, Gly-287 with Ser and Gln-432 with Arg(Rajagopal B. S. et al., Appl. Environ. Microbiol., 1998, vol.64, No.5,p. 1805-1811).

As a rule, the fbr phenotype of enzyme arises as a result of thereplacing the amino acid residue with another in a single or in a fewsites of protein sequence and these replacements lead to reducing theactivity-of enzyme. For example, the replacing of natural Met-256 with19 other amino acid residues in E. coli serine acetyltransferase (SAT)(cysE gene) leads in most cases to fbr phenotype but the mutant SATproteins do not restore the level of activity of natural SAT (NakamoriS. et al., AEM, 64(5):1607-11, 1998).

So, the disadvantage of the mutant enzymes, obtained by these methods,is a reduce in the activity of mutant enzymes as compared to wild typeenzymes.

SUMMARY OF THE INVENTION

An object of the present invention is to provide mutant feedbackresistant and high active enzymes which play a key role in biosynthesisof arginine by E. coli.

In present invention the novel procedure for synthesis a large set ofmutant argA genes is proposed by using the full randomization offragment of argA gene. The simultaneous substitutions of some amino acidresidues in fragment of protein sequence, in which the fbr mutation canbe localized, can be able to give a mutant proteins with restored thelevel of its activity near to natural due to more correct restored threedimensional structure of enzyme. Thus the present invention describedbelow has been accomplished.

That is the present invention provides:

-   -   (1) A mutant N-acetylglutamate synthase wherein the amino acid        sequence corresponding to positions from 15 to 19 in a wild type        N-acetylglutamate synthase is replaced with any one of amino        acid sequences of SEQ ID NOS: 1 to 4, and feedback inhibition by        L-arginine is desensitized;    -   (2) The mutant N-acetylglutamate synthase according to    -   (1), wherein a wild type N-acetylglutamate synthase is that of        Escherichia coli.    -   (3) The mutant N-acetylglutamate synthase according to    -   (1), which includes deletion, substitution, insertion, or        addition of one or several amino acids at one or a plurality of        positions other than positions from 15 to 19, wherein feedback        inhibition by L-arginine is desensitized;    -   (4) A DNA coding for the mutant N-acetylglutamate synthase as        defined in any one of (1) to (3);    -   (5) A bacterium belonging to the genus Escherichia which is        transformed with the DNA as defined in (4) and has an activity        to produce L-arginine; and    -   (6) A method for producing L-arginine comprising the steps of        cultivating the bacterium as defined in (5) in a medium to        produce and accumulate L-arginine in the medium and collecting        L-arginine from the medium.

The NAGS having any of fbr mutation as described above may be referredto as “the mutant NAGS”, a DNA coding for the mutant NAGS may bereferred to as “the mutant argA gene”, and a NAGS without mutation maybe referred to as “a wild type NAGS”.

Hereafter, the present invention will be explained in detail.

<1>Mutant NAGSs and Mutant ArgA Genes

The mutant NAGSs and the mutant argA genes coding the same were obtainedby randomized fragment-directed mutagenesis. To obtain the numerousmutations in argA gene the full randomization of 15-nucleotide fragmentof argA gene which codes the region from 15-th to 19-th amino acidresidues in protein sequence was carried out. The full randomized15-nucleotide fragment gives 4¹⁵ or near 10⁹ different DNA sequenceswhich can code 20⁵ different amino acid residues in 5-mer peptide. Thelikelihood of in frame non-introducing the stop codons in this sequencesis equal of about 0.95⁵ or 78%. So, the full randomization of the argAgene fragment coded the peptide from 15-th to 19-th amino acid residuesmust give approximately 2.5 million different protein sequences withdiversity in this peptide fragment of NAGS structure. Subsequentselection and screening of recombinant clones carrying mutant argA genescloned into expression vector allows to choose the fbr variants ofmutant NAGS with different level of its biological activity up to levelof activity of derepressed wildtype (wt) NAGS. In the selection, theinventors considered that the strain harboring the mutant argA genewould be obtained by using argD⁻, and proB⁻or proA⁻strain, because sucha strain cannot produce L-proline due to inhibition of NAGS therebycannot grow if excess amount of L-arginine exists in a culture medium,but the strain harboring fbr NAGS can grow in a minimal medium becauseglutamate-semialdehyde, a precursor of L-proline, can be supplied byacetylornithine deacetylase (the argE product) fromN-acetylglutamate-semialdehyde, a precursor of L-arginine (Eckhardt T.,Leisinger T., Mol. Gen. Genet., vol. 138, p.225-232, 1975). However, theinventors found that it is difficult to obtain fbr NAGS having highactivity by the above method as described in the abter-mentionedfollowing Example, and that fbr NAGS having high activity can beobtained by introducing the mutant argA into a wild type strain andselection of a strain which shows delay of cell growth.

The amino acid sequences of the mutant NAGS suitable for fbr phenotypeof NAGS were defined by the present invention. Therefore, the mutantNAGS can be obtained based on the sequences by introducing mutationsinto a wild type argA using ordinary methods. As a wild type argA gene,the argA gene of E. coli can be mentioned (GenBank Accession Y00492).

The amino acid sequence of positions from 15 to 19 in the mutant NAGS ofthe present invention is any one of the sequnece of SEQ ID NOS: 1 to 4.The corresponding amino acid sequence of known mutant NAGS, in which tyrat a position 19 is replaced with Cys, and the wild type NAGS of E. coliare illustrated in SEQ ID NOS: 5 and 6. Examples of nucleotide sequenceencoding these amino acid sequences are shown in SEQ ID NOS: 7 to 12.Table 1 shows these sequence. TABLE 1 Amino acid sequence SEQ ID NO:Nucleotide sequence SEQ ID NO: Val Val Trp Arg Ala 1 GTAGTATGGCGGGCA 7Leu Phe Gly Leu His 2 TTGTTCGGATTGCAC 8 Ser Arg Arg Ser Arg 3TCGCGGCGGTCCAGA 9 Gly Trp Pro Cys Val 4 GGGTGGCCATGCGTG 10 His Ser ValPro Cys 5 CATTCGGTTCCCTGT 11 His Ser Val Pro Tyr 6 CATTCGGTTCCCTAT 12

The mutant NAGS may including deletion, substitution, insertion, oraddition of one or several amino acids at one or a plurality ofpositions other than 15th to 19th, provided that the NAGS activity, thatis an activity to catalyze the reaction of acetylation of L-glutamicacid which produces N-acetylglutamate, is not deteriorated.

The number of “several” amino acids differs depending on the position orthe type of amino acid residues in the three dimensional structure ofthe protein. This is because of the following reason. That is, someamino acids have high homology to one another and the difference in suchan amino acid does not greatly affect the three dimensional structure ofthe protein. Therefore, the mutant NAGS of the present invention may beone which has homology of not less than 30 to 50%, preferably 50 to 70%with respect to the entire amino acid residues for constituting NAGS,and which has the fbr NAGS activity.

In the present invention, “amino acid sequence correspoding to thesequence of positions from 15 to 19” means an amino acid sequencecorresponding to the amino acid sequence of positions from 15 to 19 inthe amino acid sequence of E. coli wild type NAGS. A position of aminoacid residue may change. For example, if an amino acid residue isinserted at N-terminus portion, the amino acid residue inherentlylocates at the position 15 becomes position 16. In such a case, theamino acid residue corresponding to the original position 15 isdesignated as the amino acid residue at the position 15 in the presentinvention.

The DNA, which codes for the substantially same protein as the mutantNAGS as described above, may be obtained, for example, by modifying thenucleotide sequence, for example, by means of the site-directedmutagenesis method so that one or more amino acid residues at aspecified site involve deletion, substitution, insertion, or addition.DNA modified as described above may be obtained by the conventionallyknown mutation treatment. The mutation treatment includes a method fortreating a DNA containing the mutant argA gene in vitro, for example,with hydroxylamine, and a method for treating a microorganism, forexample, a bacterium, belonging to the genus Escherichia harboring themutant argA gene with ultraviolet irradiation or a mutating agent suchas N-methyl-N′-nitro-N-nitrosoquanidine (NTG) and nitrous acid usuallyused for the mutation treatment.

The substitution, deletion, insertion, or addition of nucleotide asdescribed above also includes mutation which naturally occurs (mutant orvariant), for example, on the basis of the individual difference or thedifference in species or genus of bacterium which harbors NAGS.

The DNA, which codes for substantially the same protein as the mutantargA gene, is obtained by expressing DNA having mutation as describedabove in an appropriate cell, and investigating NAGS activity of anexpressed product.

Also, the DNA, which codes for substantially the same protein as themutant NAGS, can be obtained by isolating a DNA which hybridizes withDNA having known argA gene sequence or a probe obtainable therefromunder stringent conditions, and which codes for a protein having theNAGS activity, from a cell harboring the mutant NAGS which is subjectedto mutation treatment.

The term “stringent conditions” referred to herein is a condition underwhich so-called specific hybrid is formed, and non-specific hybrid isnot formed. It is difficult to clearly express this condition by usingany numerical value. However, for example, the stringent conditionsinclude a condition under which DNAs having high homology, for example,DNAs having homology of not less than 50% with each other arehybridized, and DNAs having homology lower than the above with eachother are not hybridized. Alternatively, the stringent condition isexemplified by a condition under which DNA's are hybridized with eachother at a salt concentration corresponding to an ordinary condition ofwashing in Southern hybridization, i.e., 60° C., preferably 65° C.,1×SSC, 0.1% SDS, preferably 0.1×SSC, 0.1% SDS.

The gene, which is hybridizable under the condition as described above,includes those having a stop codon generated within a coding region ofthe gene, and those having no activity due to mutation of active center.However, such inconveniences can be easily removed by ligating the genewith a commercially available expression vector, and investigating NAGSactivity.

<2>Bacterium Belonging to the Genus Escherichia of the Present Invention

The bacterium belonging the genus Escherichia of the present inventionis a bacterium belonging to the genus Escherichia to which the mutantargA gene as described above is introduced. A bacterium belonging to thegenus Escherichia is exemplified by E. coli. The mutant argA gene can beintroduced by, for example, transformation of a bacterium belonging tothe genus Escherichia with a recombinant DNA comprising a vector whichfunctions in a bacterium belonging to the genus Escherihia and themutant argA gene. The mutant argA gene can be also introduced bysubstitution of argA gene on a chromosome with the mutant argA gene.

Vector using for introduction of the mutant argA gene is exemplified byplasmid vectors such as pBR322, pMW118, pUC19 or the like, phage vectorssuch as 11059, lBF101, M13mp9 or the like and transposon such as Mu,Tn10, Tn5 or the like.

The introduction of a DNA into a bacterium belonging to the genusEscherichia can be performed, for example, by a method of D. A. Morrison(Methods in Enzymology, 68, 326 (1979)) or a method in which recipientbacterial cell are treated with calcium chloride to increasepermeability of DNA (Mandel, M., and Higa, A., J. Mol. Biol., 53, 159,(1970)) and the like.

If the mutant argA gene is introduced into L-arginine-producingbacterium belonging to the genus Escherichia as described above, aproduced amount of L-arginine can be increased. Besides, an ability toproduce L-arginine may be imparted to a bacterium to which the mutantargA gene is introduced. As the bacterium belonging to the genusEscherichia which has an activity to produce L-arginine is exemplifiedby E. coli 237 strain (VKPM B-7925). The 237 strain has been depositedin Russian National Collection of Industrial Microorganisms (VKPM) underthe accession number VKPM B-7925 since Apr. 10, 2000, and transferred tothe original deposit to international deposit based on Budapest Treaty,on May 18, 2001.

<3>Method for Producing L-arginine

L-arginine can be efficiently produced by cultivating the bacterium towhich the mutant argA gene is introduced and which has an ability toproduce L-arginine, in a culture medium, producing and accumulatingL-arginine in the medium, and collecting L-arginine from the medium.

In the method of present invention, the cultivation of the bacteriumbelonging to the genus Escherichia, the collection and purification ofL-arginine from the liquid medium may be performed in a manner similarto those of the conventional method for producing L-arginine byfermentation using a bacterium. A medium used in cultivation may beeither a synthetic medium or a natural medium, so long as the mediumincludes a carbon and a nitrogen source and minerals and, if necessary,nutrients which the bacterium used requires for growth in appropriateamount. The carbon source may include various carbohydrates such asglucose and sucrose, and various organic acids, depending onassimilatory ability of the used bacterium. Alcohol including ethanoland glycerol may be used. As the nitrogen source, ammonia, variousammonium salts as ammonium sulfate, other nitrogen compounds such asamines, a natural nitrogen source such as peptone, soybean hydrolyzateand digested fermentative microbe are used. As minerals, monopotassiumphosphate, magnesium sulfate, sodium chloride, ferrous sulfate,manganese sulfate, calcium carbonate are used. The cultivation ispreferably culture under an aerobic condition such as a shaking, and anaeration and stirring culture. The temperature of culture is usually 20to 40° C., preferably 30 to 38° C. The pH of the culture is usuallybetween 5 and 9, preferably between 6.5 and 7.2. The pH of the culturecan be adjusted with ammonia, calcium carbonate, various acids, variousbases, and buffers. Usually, a 1 to 3-day cultivation leads to theaccumulation of L-arginine in the medium.

Collecting L-arginine can be performed by removing solids such as cellsfrom the medium by centrifugation or membrane filtration aftercultivation, and then collecting and purifying L-arginine by ionexchange, concentration and crystalline fraction methods and the like.

BRIEF EXPLANATION OF THE DRAWINGS

FIG. 1 shows scheme of construction of pool of mutant argA genes.

BEST MODE FOR CARRYING OUT THE INVENTION

The present invention will be specifically explained with reference tothe following examples.

EXAMPLE 1

<1>The Randomized Fragment-directed Mutagenesis

The BamHI-SalI chromosomal DNA fragment (2.02 kb) with wt argA gene wascloned into plasmid pUC19 (plasmid pUC19-ArgA). Pyrobest™ DNA Polymeraseused for PCR amplification was obtained from Takara Shuzo Co. (Japan)and is used under the conditions recommended by the supplier.

To construct the pool of mutant argA genes, at the first step thefragment of argA gene coded the sequence from 20-th amino acid residueto the end of NAGS was amplifying (FIG. 1). The plasmid pUC19-ArgA isused as the template, the sense primer P1:5′-CGAGGGATTCCGCNNNNNNNNNNNNNNNATCAATACCCACCGGG-3′(SEQ ID NO:13), isdesigned based on the nucleotide sequence of argA and the standard M13reverse sequence primer is used as a antisense primer P2. The fixed16-nucleotide 3′-end sequence of primer P1 is homologous to the sequenceof argA gene downstream Tyr-19 codon and the fixed 13′-nucleotide5′-end-to the sequence upstream His-15. The homology of 3′-end part ofP1 to argA sequence was used to synthesize the 1.75 kbp DNA fragment byusing twenty PCR cycles. 100ng of pUC19-ArgA was added as a template toPCR solution (50 μl) containing each of the two primers (40 pmol).Twenty PCR cycles (94° C. for 0.6 min, 55° C. for 0.5 min, 72° C. for 2min) is carrying out with a model 2400 DNA thermal cycler (Perkin-ElmerCo., Foster City, Calif.)

At the second step of amplification eight cycles (94° C. for 1 min, 37°C. for 1 min, 72° C. for 0.5 min) is carrying out in which the (-) chainof this fragment is functioning as a “primer” for extension it to getthe full gene sequence.

At the third step, the 10 μl aliquot of the reaction mixture is added toa fresh reaction mixture (40 μl) containing 100 pmol of the sense primerP3: 5′-TGCCATGGTAAAGGAACGTAAAACC-3′(SEQ ID NO:14), homologous to 5′-endsequence of argA gene, and primer P2 as antisense, and additional tencycles (94° C. for 0.5 min, 52° C. for 0.5 min, 72° C. for 2 min) areperformed.

The 1.78 kbp DNA fragment coding the pool of mutant variants of fulllength argA genes is purified by agarose gel electrophoresis, isdigested with NcoI (the site which includes the initial ATG codon ofargA gene) and HindIII, and then is ligated to the pKK233-2 vector(Pharmacia, Sweden) digested with NcoI and HindIII.

About 150 ng of DNA ligated is used for transformation of E. colirecipient cells in subsequent experiments to give about 2000 recombinantclones in each case.

<2>Isolation of New ArgA Mutants and Effect of Amino Acid Substitutionsin NAGS on Catalytic Properties

The plasmid vector pKK 233-2 (Pharmacia, Sweden) was used for cloningand expression of argA gene variants. The E. coli recipient strain wasTG1 (supe hsdΔ5 thi Δ(lac-proAB) F′[traD36 proAB⁺lacI^(q) lacZΔM15])(J.Sambrook et al., Molecular Cloning, 1989). The selection TG1 cellscarrying the set of recombinant plasmids pKK-argA-random (with argA genemutants) was carried out on LB agar plates. The delay of cell growth ofsome mutant clones was observed and this effect was supposed tocorrelate with production of the active fbr NAGS mutant enzymes. Theplasmids from some clones was purified and DNA sequence of 5′-fragmentsof mutant argA genes was determined by using dideoxy chain terminationmethod (table 2).

To determine the NAGS activity, the arginine auxotroph, strain E. coliB3083 (argA⁻, metB⁻) is transformed with these plasmids. The enzymes inthe soluble fractions obtained from sonicated recombinant cells ispartly purified by ammonium sulfate precipitation and assayed asdescribed below. The NAGS activity of strains carrying plasmidspKK-argA-r11 (3390 nmol/min×mg), pKK-argA-r12 (1220 nmol/min×mg) andpKK-argA-r13 (3120 nmol/min×m g) is significantly higher than the NAGSactivity of the strain harboring pKK-argA-r4 (300 nmol/min×mg). The lastplasmid carried the mutant argA gene with the same substitution (Y19C)as it was described for most active variant of argA gene with singlesubstitution by Rajagopal B. S. et al. (Rajagopal B. S. et al., Appl.Environ. Microbiol., 1998, v.64, No.5, p. 1805-1811).

Also, the activity of NAGS in strain carrying the plasmid pKK-argA(wt)(wild type argA) is lower than in the case of pKK-argA-r11, -r12 and-r13. The levels of activity of mutant enzymes are approximately thesame in presence of 10 mM arginine, while the wild-type enzyme ismarkedly inhibited by arginine (less than one-tenth). These resultsindicate that peptide fragment from 15-th to 19-th amino acid residuesis responsible for the feedback inhibition of NAGS by L-arginine and forthe level of catalytic efficiency of mutant NAGS.

(Enzyme Assay)

The acetyl coenzyme A and all chemicals used were purchased from SigmaChemical Co., St. Louis, Mo. To determinate NAGS activities, cells E.coli B3083 (argA⁻, metB⁻) carrying recombinant plasmids are grown in M9medium (5 ml) to the late exponential phase, washed with 0.14 M NaClsolution, and resuspended in 2 ml of 40 mM K-phosphate buffer (pH 7.0)with 100 mM KCl. The cells is sonicated and centrifuged. The NAGScontaining fractions are precipitated by 5 volumes of saturated(NH₄,)₂SO₄, and pellets are dissolved in 2 ml of 40 mM K-phosphatebuffer (pH 7.0) with 100 mM KCl and 30% (vol/vol) glycerol. The NAGSsolution is added to 0.1 ml of reaction mixture (100 mM tris-HCl (pH8.5), 35 mM KCl, 20 mM L-glutamate, 1.2 mM acetyl coenzyme A) andreaction mixture is incubated at 37° C. for 10 min. The reaction isstopped by adding 0.3 ml of ethanol and reaction mixture is centrifuged.0.95 ml of 0.24 mM DTNB (5,5-dithio-bis-2-nitrobenzoate) solution isadded to supernatant and mixture is incubated for 15 min. The NAGSactivity is assayed by measuring the absorbance at 412 nm.

<3>Isolation of New ArgA Mutants by Selection in B16-4 (pro⁻argD⁻) cells

The selection of mutant argA (pKK-argA-random) in E. coli B16-4 strain(pro⁻argD^(—)) was carried out by the above described procedure. Therecombinant clones from agar plates were suspended in M9 medium withL-arginine, and were grown to stationary phase. The aliquot of culturewas suspended in the fresh medium and the growth procedure was repeatedfour times. After that aliquot of culture is plated on M9 agar with 5mg/ml of L-arginine and 100 μg of ampicillin. The plasmids from someclones were purified, 5′-fragments of mutant argA genes were sequencedand the levels of activity of mutant NAGS were assayed as describedabove. The 60% of mutants carried the sequence -Gly-Trp-Pro-Cys-Val-(SEQID NO: 4) in a mutagenized fragment of enzyme and possessed a weak(about 10 nmol/min×mg) but fbr NAGS activity. Obviously, this mutantprotein provides the optimal level of NAGS activity for the growth ofpro⁻argD-cells in the selection conditions used. So, the conditions ofselection are supposed to determine the activity of the mutant NAGSobtained. TABLE 2 NAGS (ArgA) obtained by randomized fragment-directedmutagenesis Altered sequence NAGS activity Sequence of of NAGS (fragmentin the altered fragment of protein from presence of L- Clone with ofmutant argA 15-th to 19-th NAGS activity, Arg (10 Recombinant gene a.a.)nmol/min × mg* mM), %** PKKArgA-r11 GTAGTATGGCGGGCA ValValtrpArgAla 3390103% PKKArgA-r12 TTGTTCGGATTGCAC LeuPheGlyLeuHis 1220 100% PKKArgA-r13TCGCGGCGGTCCAGA SerArgArgSerArg 3120 107% PKKArgA-32- GGGTGGCCATGCGTGGlyTrpProCysVal 10.3 103% 34, 36, 38, 39 PKKArgA-r4 CATTCGGTTCCCTGTHisSerValProCys 300 91% PKKArgA-wt CATTCGGTTCCCTAT HisSerValProTyr 1200<10%*To total cellular proteins;**100% stands for activity in the absence of L-Arg.<4>Production of L-arginine by Using of Mutant ArgA Genes

The recombinant plasmids pKKArgA-r4, 11, 12, 13 and 32 were digested byBamHI and SalI, and the fragments which contained mutant argA genesunder trc promoter were sub-cloned onto low copy plasmid pMW119 (NipponGene Co., Tokyo). Resulting plasmids were designated pMADS4, pMADS11,pMADS12, pMADS13 and pMADS32, respectively. These plasmids wereintroduced into an L-arginine-producing strain E. coli 237 (VKPMB-7925). The L-arginine (Arg) and citrulline (Cit) production oftransformants are shown in Table 3. Most of the producer strains withthe new mutant NAGS'es give the higher Arg+Cit production than therecipient strain or strain with known Tyr19Cys mutant NAGS (pMADS4).TABLE 3 Production of L-arginine and citrulline. Arg Cit Arg + CitStrain (g/l) (g/l) (g/l) 237 4.7 0 4.7 237/pMADS4 8.7 0 8.7 237/pMADS1110.0 3.0 13.0 237/pMADS12 7.6 2.6 10.2 237/pMADS13 9.1 3.7 12.8237/pMADS32 8.2 0 8.2(The Cultivation Conditions in Test-tube Fermentation)

The fermentation medium contained 60 g/l glucose, 25 g/l ammoniasulfate, 2 g/l KH₂PO₄, 1 g/l MgSO₄, 0.1 mg/l thiamine, 5 g/l yeastextract Difco, 25 g/l chalk, per 1 liter of tap water (pH 7.2). Glucoseand chalk were sterilized separately. 2 ml of the medium was placed intotest-tubes, inoculated with one loop of the tested microorganisms, andthe cultivation was carried out at 32° C. for 3 days with shaking.

1.-3. (Canceled)
 4. A DNA coding for the mutant N-acetylglutamatesynthase wherein amino acid residues corresponding to positions 15 to 19in the N-acetylgluatmate synthase of SEQ ID NO: 16 are replaced with anyone of amino acid sequences of SEQ ID NOS: 1 to 4, and feedbackinhibition by L-arginine is desensitized wherein the N-acetylglutamatesynthase is a protein defined in the following (A) or (B): (A) a proteinhaving an amino acid sequence defined in SEQ ID NO: 16; or (B) a proteinthat is encoded by a DNA which hybridizes with a DNA having thenucleotide sequence defined in SEQ ID NO: 15 under stringent conditions,wherein said stringent conditions entail a temperature ranging from 60°C. to 65° C., a salt concentration ranging from 0.1×SSC to 1×SSC, and0.1% SDS, and wherein the mutant of N-acetylglutamate synthase has aN-acetylglutamate synthase activity.
 5. A bacterium belonging to thegenus Escherichia which is transformed with the DNA as defined in claim4 and has an activity to produce L-arginine.
 6. A method for producingL-arginine comprising the steps of cultivating the bacterium as definedin claim 5 in a medium to produce and accumulate L-arginine in themedium and collecting L-arginine from the medium.
 7. The DNA as definedin claim 4, wherein the N-acetylglutamate synthase has an amino acidsequence defined in SEQ ID NO:
 16. 8. The DNA as defined in claim 7,wherein positions 15 to 19 are replaced with the amino acid sequences ofSEQ ID NO:
 1. 9. The DNA as defined in claim 7, wherein positions 15 to19 are replaced with the amino acid sequences of SEQ ID NO:
 2. 10. TheDNA as defined in claim 7, wherein positions 15 to 19 are replaced withthe amino acid sequences of SEQ ID NO:
 3. 11. The DNA as defined inclaim 7, wherein positions 15 to 19 are replaced with the amino acidsequences of SEQ ID NO:
 4. 12. The DNA as defined in claim 4, whereinthe N-acetylglutamate synthase is a protein that is encoded by a DNAwhich hybridizes with a DNA having the nucleotide sequence defined inSEQ ID NO: 15 under stringent conditions, wherein said stringentconditions entail a temperature ranging from 60° C. to 65° C., a saltconcentration ranging from 0.1×SSC to 1×SSC, and 0.1% SDS, and whereinsaid protein has a N-acetylglutamate synthase activity.
 13. The DNA asdefined in claim 12, wherein positions 15 to 19 are replaced with theamino acid sequences of SEQ ID NO:
 1. 14. The DNA as defined in claim12, wherein positions 15 to 19 are replaced with the amino acidsequences of SEQ ID NO:
 2. 15. The DNA as defined in claim 12, whereinpositions 15 to 19 are replaced with the amino acid sequences of SEQ IDNO:
 3. 16. The DNA as defined in claim 12, wherein positions 15 to 19are replaced with the amino acid sequences of SEQ ID NO: 4.